While working on the preservation of some of my bacterial cultures i have unfortunately missed the steps of incubating the inoculum suspended in 500 ul of nutrient broth for overnight and did not mix it after the addition of glycerol. Are the bacterial cultures preserved going to work or have i lost them completely?
Dear Abdul Rauf Tareen: You have missed the crucial and essential step for preservation of microorganism; overnight growth is needed to expand the culture. You might no longer able to gain your original culture.
From what you wrote, it sounds like to picked a colony, mixed it in nutrient broth, then added glycerol and froze? If so, yeah, I don't think those cultures will be very good. The overnight growth is needed to expand the culture, and the glycerol is needed to protect it.
How about double picking the colony? Try inoculating again with the same colony, and you'll find out tomorrow if it worked!
If you can't double pick, you could also thaw the entire glycerol stock and dump it on a plate- hopefully a few bacterium survived and you could start again, but this would probably be a rather messy test with the glycerol.
The overnight incubation allows the bacteria to proliferate and attain higher number. Mixing it with glycerol helps to avoid contamination. If you picked a colony without incubation, that's not so good as it reduces the chances of recovering viable bacteria.
However, it's a bargain. You can thaw the samples and try to recover the bacteria. You may be lucky
Dear Abdul Rauf Tareen: You have missed the crucial and essential step for preservation of microorganism; overnight growth is needed to expand the culture. You might no longer able to gain your original culture.
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